title: Analysis of protein aggregation kinetics using short amino acid peptide tags.
abstract: Understanding protein solubility, and consequently aggregation, is an important issue both from an academic and a biotechnological application viewpoints. Here we report the effects of 10 representative amino acids on the aggregation kinetics of proteins. The effects were determined by measuring the solubility of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, to which short artificial tags containing the amino acid of interest were added at its C-terminus. We determined the solubility of the tagged variants as a function of equilibration time (20 min to 48 h) and total protein concentration ranging from 0.10mg/ml to 25.0mg/ml. We observed, as anticipated, that proteins precipitated when the total protein concentration exceeded a critical value. However, when the total protein concentration was further increased, the apparent solubility reached a concentration above the critical value, and slowly decreased to a value under the critical concentration upon increasing the equilibration period. We rationalized these observations by identifying three different solubility values, the "transient solubility (TS)", the "aggregation initiation concentration (AIC)" and the "long-term solubility (LS)". AIC and LS are parameters determined essentially by the amino acid types composing the tags and could be considered as an amino acid's intrinsic property. On the other hand, TS is an apparent solubility that is measured after some (20 min in our case) equilibration time and is often considered as the "solubility" of the protein. Similar aggregation kinetic patterns were observed with natural proteins, indicating the generality of the observations made using our model protein.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.
authors.forename: Monsur Alam
authors.initials: MA
authors.lastname: Khan
authors.forename: Mohammad Monirul
authors.initials: MM
authors.lastname: Islam
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kuroda
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D007611:Aprotinin / Q000737:chemistry*; D002417:Cattle; D011232:Chemical Precipitation; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D013194:Staining and Labeling; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D007611:Aprotinin
keywords: AIC; Aggregation kinetics; BPTI; BPTI-19; Equilibration time; LS; Poly amino acid peptide tag; Protein aggregation; Protein solubility; R; TS; aggregation initiation concentration; bovine pancreatic trypsin inhibitor; correlation coefficient; in vitro amorphous protein aggregation; long-term solubility; simplified single-disulfide-bonded BPTI variant containing 19 alanines; transient solubility